Prolonged ethanol exposure causes central nervous system hyperexcitability that involves a loss of GABAergic inhibition. We previously demonstrated that long-term ethanol exposure enhances the internalization of synaptic GABA A receptors composed of ␣1␤2/3␥2 subunits. However, the mechanisms of ethanol-mediated internalization are unknown. This study explored the effect of ethanol on surface expression of GABA A ␣1 subunit-containing receptors in cultured cerebral cortical neurons and the role of protein kinase C (PKC) ␤, ␥, and isoforms in their trafficking. Cultured neurons were prepared from rat pups on postnatal day 1 and maintained for 18 days. Cells were exposed to ethanol, and surface receptors were isolated by biotinylation and P2 fractionation, whereas functional analysis was conducted by whole-cell patch-clamp recording of GABAand zolpidem-evoked responses. Ethanol exposure for 4 h decreased biotinylated surface expression of GABA A receptor ␣1 subunits and reduced zolpidem (100 nM) enhancement of GABA-evoked currents. The PKC activator phorbol-12,13-dibutyrate mimicked the effect of ethanol, and the selective PKC inhibitor calphostin C prevented ethanol-induced internalization of these receptors. Ethanol exposure for 4 h also increased the colocalization and coimmunoprecipitation of PKC␥ with ␣1 subunits, whereas PKC␤/␣1 association and PKC/␣1 colocalization were not altered by ethanol exposure. Selective PKC␥ inhibition by transfection of selective PKC␥ small interfering RNAs blocked ethanol-induced internalization of GABA A receptor ␣1 subunits, whereas PKC␤ inhibition using pseudo-PKC␤ had no effect. These findings suggest that ethanol exposure selectively alters PKC␥ translocation to GABA A receptors and PKC␥ regulates GABA A ␣1 receptor trafficking after ethanol exposure.

All currently efficacious antipsychotic drugs have as part of their mechanism the ability to attenuate some or all of their signaling through the dopamine D2 receptor. More recently, the dopamine D1 receptor has been hypothesized to be a promising target for the treatment of negative and/or cognitive aspects of schizophrenia that are not improved by current antipsychotics. Although cAMP has been presumed to be the primary messenger for signaling through the dopamine receptors, the last decade has unveiled a complexity that has provided exciting avenues for the future discovery of antipsychotic drugs (APDs). We review the signaling mechanisms of currently approved APDs at dopamine D2 receptors, and note that aripiprazole is a compound that is clearly differentiated from other approved drugs. Although aripiprazole has been postulated to cause dopamine stabilization due to its partial D2 agonist properties, a body of literature suggests that an alternate mechanism, functional selectivity, is of primary importance. Finally, we review the signaling at dopamine D1 receptors, and the idea that drugs that activate D1 receptors may have use as APDs for improving negative and cognitive symptoms. We address the current state of drug discovery in the D1 area, and its relationship to novel signaling mechanisms. Our conclusion is that although the first APD targeting dopamine receptors was discovered more than a half-century ago, recent research advances offer the possibility that novel and/or improved drugs will emerge in the next decade.

J. Neurochem. (2010) 112, 784–796. Abstract The mechanisms of ethanol actions that produce its behavioral sequelae involve the synthesis of potent GABAergic neuroactive steroids, specifically the GABAergic metabolites of progesterone, (3α,5α)‐3‐hydroxypregnan‐20‐one (3α,5α‐THP), and deoxycorticosterone, (3α,5α)‐3,21‐dihydroxypregnan‐20‐one. We investigated the mechanisms that underlie the effect of ethanol on adrenal steroidogenesis. We found that ethanol effects on plasma pregnenolone, progesterone, 3α,5α‐THP and cortical 3α,5α‐THP are highly correlated, exhibit a threshold of 1.5 g/kg, but show no dose dependence. Ethanol increases plasma adrenocorticotropic hormone (ACTH), adrenal steroidogenic acute regulatory protein (StAR), and adrenal StAR phosphorylation, but does not alter levels of other adrenal cholesterol transporters. The inhibition of ACTH release, de novo adrenal StAR synthesis or cytochrome P450 side chain cleavage activity prevents ethanol‐induced increases in GABAergic steroids in plasma and brain. ACTH release and de novo StAR synthesis are independently regulated following ethanol administration and both are necessary, but not sufficient, for ethanol‐induced elevation of plasma and brain neuroactive steroids. As GABAergic steroids contribute to ethanol actions and ethanol sensitivity, the mechanisms of this effect of ethanol may be important factors that contribute to the behavioral actions of ethanol and risk for alcohol abuse disorders.

SKF-83959 [6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine] is reported to be a functionally selective dopamine D1 receptor ligand with high bias for D1-mediated phospholipase C (PLC) versus D1-coupled adenylate cyclase signaling. This signaling bias is proposed to explain behavioral activity in both rat and primate Parkinson’s disease models, and a D1-D2 heterodimer has been proposed as the underlying mechanism. We have conducted an in-depth pharmacological characterization of this compound in dopamine D1 and D2 receptors in both rat brain and heterologous systems expressing human D1 or D2 receptors. Contrary to common assumptions, SKF-83959 is similar to the classical, well-characterized partial agonist SKF38393 in all systems. It is a partial agonist (not an antagonist) at adenylate cyclase in vitro and ex vivo, and is a partial agonist in D1-mediated β-arrestin recruitment. Contrary to earlier reports, it does not have D1-mediated effects on PLC signaling in heterologous systems. Because drug metabolites can also contribute, its 3-N-demethylated analog also was synthesized and tested. As expected from the known structure-activity relationships of the benzazepines, this compound also had high affinity for the D1 receptor and somewhat higher intrinsic activity than the parent ligand, and also might contribute to in vivo effects of SKF-83959. Together, these data demonstrate that SKF-83959 is not a highly-biased functionally selective D1 ligand, and that its reported behavioral data can be explained solely by its partial D1 agonism in canonical signaling pathway(s). Mechanisms that have been proposed based on the purported signaling novelty of SKF-83959 at PLC should be reconsidered.